Time-delay integration in electrophoretic detection systems

ABSTRACT

An apparatus for detecting analytes in a sample is provided. The apparatus includes: one or more channels having a detection zone; one or more irradiation sources disposed for irradiating the detection zone with non-coherent radiation; a detector array disposed for collecting light signals emitted from markers in the detection zone excited by the radiation, the detector array having an output; and a system coupled to the detector array for effecting time delay integration of the charges on the detector array corresponding to the light signals by accumulating the charges before reading the charges at the output of the detector array. Other apparatus and methods for detecting analytes in a sample are also provided.

PRIORITY AND RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/205,028, filed Jul. 25, 2002, which is incorporated hereinin its entirety by reference.

FIELD

The various embodiments relate to electrophoretic detection systems,and, in particular, to arrangements and methods for light detectionduring electrophoresis.

INTRODUCTION

Well-known examples of biopolymer analysis using DNA sequencing aretaught, for example, in F. Sanger, et. al., DNA Sequencing with ChainTerminating Inhibitors, 74 Proc. Nat. Acad. Sci. USA 5463 (1977); LloydM. Smith, et. al., Fluorescence detection in automated DNA sequenceanalysis, 321 Nature 674 (1986); Lloyd M. Smith, The Future of DNASequencing, 262 Science 530 (1993). These and all other publications andpatents cited herein are incorporated herein in their entireties byreference.

Known capillary electrophoresis systems and methods can requirelaser-induced fluorescence for exciting marker compounds or markers,such as dye molecules typically used to label analytes. Unfortunately,laser apparatuses are expensive to use, energy inefficient, consumelarge amounts of power, and are physically large, renderingelectrophoretic systems incorporating lasers cumbersome to use.

The use of sources of irradiation other than lasers for the excitationof marker compounds provides many advantages. Although the use of lightemitting diodes (LED's) for generating fluorescence in dye molecules istaught, for example, in U.S. Pat. Nos. 6,005,663 and 5,710,628, thecontents of which are incorporated herein in their entireties byreference, the use of LED's in such electrophoretic methods typicallyresults in low signal strengths and detection sensitivity that is onlymarginal. The low signal strength tends to impair adequate detection ofmarker compounds.

Electrophoresis arrangements that employ complex optics causing areduction of the collecting efficiency of the detector also result inlow sensitivity. The low sensitivity is aggravated through the use ofLED's, which compromise sensitivity in the first instance. The problemof lowered sensitivity through LED use is further exacerbated when usingmultiple-channel-type electrophoretic devices, that is, electrophoreticdevices using an etched plate with capillary-sized grooves, or using aplurality of capillary tubes, where either type of device hasmultiple-channels used to increase throughput.

An electrophoretic apparatus and method that include a cost-effectiveand convenient source of irradiation and that do not compromisesensitivity or resolution would be desirable, especially inmultiple-channel electrophoretic systems used to increase throughput.

SUMMARY

According to various embodiments, the present invention can relate to anapparatus and method for detecting components, for example, analytes, ina sample.

According to various embodiments, an apparatus is provided for detectinganalytes in a sample. According to various embodiments, the apparatuscan include: one or more channels having a detection zone; one or moreirradiation sources disposed for irradiating the detection zone withnon-coherent radiation; a detector array disposed for collecting lightsignals emitted from markers in the detection zone excited by theradiation, where the detector array can have an output; and a systemcoupled to the detector array for effecting time delay integration ofthe charges on the detector array corresponding to the light signals byaccumulating the charges before reading the charges at the output of thedetector array.

According to various embodiments, an apparatus is provided for detectinganalytes in a sample, wherein the apparatus can include: one or morechannels having a detection zone; one or more irradiation sourcesdisposed for irradiating the detection zone with radiation; a detectorarray disposed for collecting light signals emitted from markers in thedetection zone excited by the radiation, where the detector array canhave an output; and a system coupled to the detector array for effectingtime delay integration of the charges on the detector arraycorresponding to the light signals by accumulating the charges beforereading the charges at the output of the detector array, wherein thesystem for effecting time delay integration can do so by moving,relative to one another, the detector array and light signals from thedetection zone.

According to various embodiments, an apparatus for detecting analytes ina sample is provided, wherein the apparatus can include: one or morechannels having a detection zone; one or more irradiation sourcesdisposed for irradiating the detection zone with radiation; a detectorarray disposed for collecting light signals emitted from markers in thedetection zone excited by the radiation, where the detector array canhave an output; a system coupled to the detector array for effectingtime delay integration of the charges on the detector arraycorresponding to the light signals by accumulating the charges beforereading the charges at the output of the detector array; and are-imaging lens disposed between the detection zone and the detectorarray for optically inverting an image produced by the light signalsbefore the image is collected by the detector array.

According to various embodiments, an apparatus for detecting analytes ina sample is provided wherein the apparatus can comprise: achannel-defining member defining at least one channel therein having adetection zone; and a separating system coupled to the at least onechannel for separating a sample containing analytes and disposed incontact with a migration medium disposed within the at least one channelinto analyte bands migrating along the at least one channel, whereineach analyte band can be detectable by the presence of a correspondingmarker. According to various embodiments, the apparatus can furtherinclude: at least one irradiation source for emitting non-coherentradiation and disposed for irradiating the detection zone of the atleast one channel to thereby excite markers responsive to the radiationand which emit light signals indicative of corresponding analytes; adetector array disposed for collecting the light signals produced by themarkers and for producing charges corresponding to the light signals,where the detector array can have an output; modulating optics formodulating light between the irradiation source and the detector array;and a time delay integration system for effecting, within the detectorarray, an accumulation of charges corresponding to light signalsassociated with at least one given analyte band before readingaccumulated charges at the output of the detector array, theaccumulation being effected during an integration time of the at leastone given analyte band moving across the detection zone.

According to various embodiments, an apparatus is provided for detectinganalytes in a sample, wherein the apparatus can include: achannel-defining member defining at least one channel therein having adetection zone; and a separating system coupled to the at least onechannel for separating a sample containing analytes and disposed incontact with a migration medium in the at least one channel into analytebands migrating along the at least one channel, wherein each analyteband can be detectable by the presence of a corresponding marker.According to various embodiments, the apparatus can further include atleast one irradiation source disposed for irradiating the detection zoneof the at least one channel with radiation to thereby excite markersresponsive to the radiation and which emit light signals indicative ofcorresponding analytes; a detector array disposed for collecting thelight signals produced by the markers and for producing chargescorresponding to the light signals, where the detector array can have anoutput; modulating optics for modulating light between the at least oneirradiation source and the detector array; and a time delay integrationsystem for effecting, within the detector array, an accumulation ofcharges corresponding to light signals associated with at least onegiven analyte band before reading accumulated charges at the output ofthe detector array. The accumulation of charges can be effected duringan integration time of the at least one given analyte band moving acrossthe detection zone, by, for example, moving, relative to one another,the detector array and at least one of the detection zone and themodulating optics.

According to various embodiments, an apparatus is provided for detectinganalytes in a sample, wherein the apparatus can include: achannel-defining member defining at least one channel therein having adetection zone; and a separating system coupled to the at least onechannel for separating a sample containing analytes and disposed incontact with a migration medium in the at least one channel into analytebands migrating along the at least one channel, wherein each analyteband can be detectable by the presence of a corresponding marker. Theapparatus can further include at least one irradiation source disposedfor irradiating the detection zone of the at least one channel withradiation to thereby excite markers responsive to the radiation foremitting light signals indicative of corresponding analytes. Together,the light signals can form an image corresponding to analyte bandsmigrating across the detection zone. The apparatus can also include adetector array disposed for collecting the light signals produced by themarkers and for producing charges corresponding to the light signals,the detector array having an output; a re-imaging optical systemdisposed between the detection zone and the detector array for opticallyinverting an image produced by the light signals before the image iscollected by the detector array; modulating optics for modulating lightbetween the at least one irradiation source and the detector array; anda time delay integration system for effecting, within the detectorarray, an accumulation of charges corresponding to light signalsassociated with at least one given analyte band before readingaccumulated charges at the output of the detector array, theaccumulation being effected during an integration time of the at leastone given analyte band moving across the detection zone.

Various embodiments can pertain to a method for detecting analytes in asample, and can comprise the steps of: providing a channel-definingmember defining at least one channel therein having a detection zone;providing migration medium within the at least one channel; separating asample containing analytes and disposed in contact with the migrationmedium into analyte bands migrating along the at least one channel,wherein each analyte band can be detectable by the presence of a marker;irradiating the detection zone using at least one irradiation sourceproviding non-coherent radiation that can thereby excite markersresponsive to the radiation and that can emit light signals indicativeof corresponding analytes; detecting the light signals produced by themarkers by, for example, collecting the light signals on a detectorarray to produce charges on the detector array corresponding to thelight signals; modulating light between the at least one irradiationsource and the detector array; effecting a time delay integration of thelight signals within the detector array by, for example, accumulatingthe charges within the detector array corresponding to light signalsassociated with at least one given analyte band during an integrationtime of the at least one given analyte band moving across the detectionzone; and reading the accumulated charges.

Furthermore, according to various embodiments, an apparatus fordetecting analytes in a sample is provided that can comprise: meansdefining at least one channel therein having a detection zone; means forseparating a sample containing analytes and disposed in contact with amigration medium disposed within the at least one channel into analytebands migrating along the at least one channel, wherein each analyteband can be detectable by the presence of a marker; means forirradiating the detection zone with non-coherent radiation, that canthereby excite markers responsive to the radiation and that can emitlight signals indicative of corresponding analytes; means for detectingthe light signals by collecting the light signals that can therebyproduce charges corresponding thereto; means for effecting a time delayintegration of the light signals within the detector array by, forexample, accumulating within the detector array the chargescorresponding to light signals associated with at least one givenanalyte band during an integration time of the at least one givenanalyte band moving across the detection zone; and means for reading theaccumulated charges.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only.The accompanying drawings, which are incorporated in and constitute apart of this application, illustrate several exemplary embodiments andtogether with the instant description, serve to explain the principlesof the present invention.

DRAWINGS

The skilled artisan will understand that the drawings described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 is a schematic, side-elevational view of an electrophoresisarrangement according to various embodiments showing a channel-definingmember in cross-section;

FIG. 2 is a schematic view of the image produced on the detector arrayof a detector at a time t using the arrangement of FIG. 1;

FIG. 3 is a view similar to FIG. 2 showing the image at a time t+Δt;

FIG. 4 is a schematic, front-elevational view of an electrophoresisarrangement according to various embodiments for the sequential use ofmultiple-color irradiation sources along with filters on a filter wheel;

FIG. 5 a is a schematic, top-plan view of the arrangement of FIG. 4;

FIG. 5 b is a schematic, side-elevational view of the arrangement ofFIG. 4;

FIGS. 6 a through 6 e are respective schematic views of images producedon the detector array of a detector, each image corresponding to lightsignals filtered through a respective filter on the filter wheel of FIG.4;

FIG. 6 f is a schematic representation of an electropherogram showingfluorescence intensity curves for each filter of the filter wheel inFIG. 4 during three signal readings by the detector;

FIG. 6 g is a schematic representation of the intensity curves of FIG. 6f in aligned format for multicomponenting;

FIG. 6 h is a schematic representation of multicomponented intensitycurves for five different kinds of markers that can be used in thesystem of FIG. 4 based on the readings shown in FIG. 6 f;

FIG. 6 i is a graph of excitation efficiency versus wavelength for fourexemplary markers;

FIG. 6 j is a graph of fluorescence intensity versus wavelength for theexemplary markers of FIG. 6 i;

FIG. 7 is a top-plan, partially cross-sectional view of anelectrophoresis arrangement according to various embodiments for thesequential use of multiple-color irradiation sources along with filterson a filter wheel and along with an offset system;

FIG. 8 is a schematic view of an image produced on the detector array ofa detector using the arrangement of FIG. 7;

FIG. 9 a is a graph of relative excitation intensity versus wavelengthfor a pair of LED's used in yet another embodiment of the presentinvention, the LED's being of different colors and being used toirradiate the detection zone simultaneously;

FIG. 9 b is a graph showing percent transmission versus wavelength for aconditioning filter used to condition the light from the LED's of FIG. 9a;

FIG. 9 c is a graph showing percent transmission versus wavelength for abandpass filter used to filter through light signals produced by markersexcited by the light from the LED's of FIG. 9 a;

FIG. 9 d is a graph showing relative emission intensity versuswavelength for the light filtered through the bandpass filter of FIG. 9c;

FIG. 10 is a schematic, top-plan view of an irradiation zone showingthree channels having been selectively masked to present respectivewindows according to another embodiment of the present invention;

FIG. 11 is a schematic view of a detector array, the detector arrayhaving been separated into respective frames for use with light signalsemitted from the respective windows of the channels in FIG. 10;

FIG. 12 a is a partially cut away view of one of the frames of thedetector array shown in FIG. 11; and

FIG. 12 b is a graph showing percent integration per pixel throughoutthe width of the frame in shown in FIG. 12 a.

DESCRIPTION

The apparatuses and methods herein can address the need for anelectrophoresis device and process that employs a cost-effective andconvenient source of irradiation. Various embodiments can be especiallywell suited for multiple-channel electrophoretic systems used toincrease throughput.

FIG. 1 shows an exemplary embodiment of an electrophoresis device. Asdepicted in FIG. 1, the arrangement can include a channel-definingmember 10 defining a channel 12 therein for the migration of an analytesample. The channel-defining member 10 may include a cover plate with orwithout grooves, an etched plate defining one or more capillary sizedgrooves therein, or one or more capillary tubes. According to variousembodiments, the channel-defining member can be an etched plate having aplurality of channels or grooves, or the channel-defining member caninclude a plurality of capillary tubes. The use of a plurality ofchannels can allow a large number of analyte samples to be measuredsimultaneously in order to increase throughput. As is well known, forelectrophoresis to occur, opposing ends of channel-defining member 10,such as an electrophoretic plate or capillary tube, can be placed incontact with corresponding electrodes connected to a power supply forgenerating an electric field across the plate or tube. This field cancause the analyte to migrate from a loading site (not shown) for theplate or tube arrangement of the channel-defining member 10, toward adetection site or detection zone 14. The detection zone can encompassthat zone on the channel that is irradiated by an irradiation source toexcite markers, such as dye markers, used to label analytes in thesample.

An example of a marker compound is a dye marker. Any suitable marker,such as, for example, a fluorophore, can be used. Fluorophores usefulaccording various embodiments can include those that can be coupled toorganic molecules, particularly proteins and nucleic acids, and that canemit a detectable amount of radiation or light signal in response toexcitation by an available excitation source. Suitable markers canencompass materials having fluorescent, phosphorescent, and/or otherelectromagnetic radiation emissions. Irradiation of the markers cancause them to emit light at varying frequencies depending on the type ofmarker used.

One class of markers provides signals for the detection of labeledextension and amplification products by fluorescence, chemiluminescence,and electrochemical luminescence (Kricka, L. in Nonisotopic DNA ProbeTechniques, Academic Press, San Diego, pp. 3-28 (1992)).Chemiluminescent labels include 1,2-dioxetane compounds (U.S. Pat. No.4,931,223; and Bronstein, Anal. Biochemistry 219:169-81 (1994)).Fluorescent dyes useful for labeling probes, primers, and nucleotide5′-triphosphates include fluoresceins, rhodamines (U.S. Pat. Nos.5,366,860; 5,936,087; and 6,051,719), cyanines (Kubista, WO 97/45539),and metal porphyrin complexes (Stanton, WO 88/04777). Fluorescentreporter dyes include xanthene compounds such as fluoresceins I andrhodamines II:

The ring positions of I and II above may be substituted. The amino Rgroups of II may be substituted. The substituents can include covalentattachments to the primers, probes and nucleotides. Examples of I and IIformulae include wherein X can be phenyl substituted with carboxyl,chloro, and other groups, for example, as described in U.S. Pat. Nos.5,847,162; 6,025,505; 5,674,442; 5,188,934; 5,885,778; 6,008,379;6,020,481; and 5,936,087, which are incorporated herein in theirentireties by reference, and wherein X can be hydrogen, for example, asdescribed in U.S. Pat. No. 6,051,719, which is incorporated herein inits entirety by reference.

In the embodiment shown in FIG. 1, an irradiation source is providedthat emits non-coherent light in a given frequency range, such as, forexample, a light emitting diode (LED) 16. It is to be noted that, in theinstant description, a source of non-coherent light can be a sourceemitting light that does not encompass laser light. According to variousembodiments, the non-coherent light can have a frequency of about 660 nmor lower.

According to various embodiments, the light from the LED can bemodulated by an excitation modulating optics system before reaching thedetector zone 14. The excitation modulating optics system can include,as shown, a conditioning filter 18, the role of which can be tosubstantially block predetermined ranges of wavelengths of light emittedby the LED. The predetermined ranges can correspond to wavelengths oflight that can overlap with the emission spectra of the markers beingused. According to various embodiments, the conditioning filter may letthrough only light in the wavelength range of excitation light for oneor more of the markers. Typically, any given LED emits excitation lightin a spectral range. The range of wavelengths of the excitation light inturn may typically excite markers to emit light signals within a givenspectral range in the detection zone. For the detection of light signalsfrom the detection zone, one can block out that portion of theexcitation light that would be in the same wavelength range as some orall of the light signals emitted from the detection zone. Otherwise, itcan be difficult to determine which portion of the detected light ismerely excitation light from the LED. The light passing through theconditioning filter 18 is conditioned light 20, as seen in FIG. 1. Theexcitation modulating optic can further include a focusing opticalsystem 19. The conditioned light 20 can thereafter be focused byfocusing optical system 19 for irradiating the analyte sample andcorresponding markers in the detection zone 14. The thus irradiatedmarker or markers in turn emit light signals, such as throughfluorescence, at frequencies specific to the irradiated marker, so as topresent a peak intensity. For example, a dye excited by yellow lightmight have a fluorescence emission peak intensity at 610 nmcorresponding to the orange portion of the spectrum, while a peak atabout 460 nm is associated with the blue portion of the spectrum, and apeak at about 660 nm is associated with the red portion of the spectrum.It is noted that there are only a limited number of colors possible forefficient laser irradiation sources when compared with possible colorsfor efficient, non-coherent irradiation sources, such as LED's. The useof irradiation sources emitting non-coherent radiation in turn allowsthe use of a wider range of markers when compared with the use oflasers. By way of example, ROX, a known dye marker, is best excited at590 nm. No laser, however, is particularly efficient at 590 nm. ROX isbetter excited by an LED emitting radiation at 590 nm.

The device of FIG. 1 further includes a collection modulating opticssystem that can include a collimating optical system 24, a wide bandpassfilter 26, a transmission grating 28, and a re-imaging optical system30. Emitted light 22 from the detection zone 14, and, in addition,conditioned light 20 passing through the detection zone 14, arecollimated by a first optical component or system 24. In this respect,it is to be noted that the light from the detection zone includes theemitted light 22 and a portion of the conditioned light 20 passingthrough the detection zone. Alternatively, the excitation light can bebrought in at an angle with respect to the detection zone such that mostof the conditioned light passing through the detection zone is notcollected by the collimating optical system 24. This reduces theexcitation light that must be rejected. However, such an alternativearrangement also decreases the level of excitation light that hits thedetection zone 14. It is, nevertheless, possible to establish acompromise between irradiation angle and level of excitation light, asreadily recognizable by one skilled in the art.

The light 20 and 22 from the detection zone 14 can be collimated bycollimating optical system 24. By collimation, what is meant in thecontext of various embodiments is any reduction in the propagation angleof the light being collimated. According to various embodiments, thereduction in the propagation angle of the light being collimated canresult in a propagation angle between about 20 degrees and about 0degrees.

According to various embodiments, a long pass filter, or, in thealternative, a wide bandpass filter, indicated by reference numeral 26,can thereafter be used for letting through, substantially exclusively,predetermined wavelengths of light from the detection zone correspondingto a portion of wavelengths of the light signals emitted by anassociated marker. The portion of the wavelengths of the light signalscan include, for example, all of the light signals, or it can include,for each marker, a range of wavelengths of the light signals, such as arange of wavelengths about the peak intensity of the light signals. Asan example, a wide bandpass filter can block wavelengths of lightoutside of the range of from about 500 nm to about 700 nm, therebyletting through only light that corresponds very specifically to thelight emitted by the markers, that is, corresponding to emitted light22. Thereafter, emitted light 22 can be spectrally distributed by atransmission grating 28 and refocused by re-imaging optical system 30onto an array 34 of solid-state detectors, or detector array 34. Theexcitation modulating optics system and the collection modulating opticssystem are hereinafter collectively referred to as “the modulatingoptics system” or “modulating optics”. According to various embodiments,the array of solid-state detectors can include the photo-detectingsurface of the parallel register of a charge-coupled device (CCD) 32. Inthe shown embodiment, the image produced by a refocusing of emittedlight 22 is projected onto the detector array 34 of the CCD, producing apattern of charge in proportion to the total integrated flux incident oneach pixel of the parallel register, as is well known in the art.

Referring additionally now to FIGS. 2 and 3, an image produced by movinganalyte bands is recorded by the photo-detecting surface of CCD 32(FIG. 1) at times t (FIG. 2) and t+Δt (FIG. 3). The photo-detectingsurface 36 can be, according to an embodiment, part of the twodimensional detector array 34 shown above with respect to FIG. 1.Photo-detecting surface 36 can include a spectral axis as indicated byarrow λ on the figure, and a spatial axis along which the analyte bandsmove, as indicated by arrow M in FIGS. 2 and 3. As further seen in FIG.2, the image created by the light signals emitted by excited markersproduces two bands 38 and 40 on photo-detecting surface 36 substantiallyin the red and blue regions of the spectrum, respectively, each bandcorresponding to a marker used to label, for example, a predeterminedtype of analyte. The bands are spectrally distributed along the spectralaxis by transmission grating 28 in FIG. 1. At time t, as shown in FIG.2, the charges produced on surface 36 present two respective peaks 46and 48 on intensity profile 45. These peaks correspond to bands 38 and40, respectively. As seen in FIG. 3, at time t+Δt, both bands 38 and 40have moved downward along the direction of migration M on thephoto-detecting surface 36. Serial register 42 of the CCD 32 (FIG. 1)collects the charges accumulated for each analyte band during itsintegration time. All signals received from the detector can beconverted from analog to digital and conveyed to a serial port fortransmission to a multipurpose computer for storage and for furtherprocessing and analysis. The analog output can alternatively oradditionally be sent directly to an output device for display orprinting, or used for other purposes.

According to various embodiments, for example, as shown in theembodiment above, collection of the image is performed using time delayintegration (TDI). In the CCD, the photogenerated charge in thephotoactive elements or pixels can be transferred toward the serialregister 42 one row at a time. The charge information in the serial rowis then read by using a corresponding single on-chip amplifier orreadout register 44 of the CCD. By way of example, for a 256×256 elementCCD, each time a single imaging area is transferred to the serialregister 42, 256 readouts of the thus transferred area are performed,each readout corresponding to a different spectral element. The aboveprocess continues until all 256 rows have been read 256 times.

Under a normal read-out approach, the motion of the images on thedetector array 34 produces a blur. In TDI, according to variousembodiments, the shutter is eliminated and the shifting of rows of theCCD is synchronized to the migration of the band of analyte in thechannel. Thus, as an analyte band enters the excitation zone of the LED(detection zone 14), the light signals emitted therefrom are collectedand illuminate the first row of the CCD, and the charge information isread using the CCD's amplifier or readout register 44. The band takes aperiod of time, Δtp, to migrate on the channel so that its imagemigrates to the next row of the CCD, one row closer to the serialregister. After this time period, the charge on the CCD can be shiftedone row closer to the serial register, such that the fluorescence fromthe analyte still corresponds to the same charge information on the CCD.Therefore, distinct from the physical rows of the CCD, there exists inTDI according to various embodiments a continuously moving row ofaccumulating photogenerated charge. An example of TDI in a CE systemusing LIF is disclosed in U.S. Pat. No. 5,141,609 to Sweedler et al.,and in J. F. Sweedler et al., Fluorescence Detection in Capillary ZoneElectrophoresis Using a Charge-Coupled Device with Time DelayedIntegration, Anal. Chem. 63, 496-502 (1991), the contents of both ofwhich are incorporated herein in their entireties by reference. Theeffective integration time for a given analyte band can vary accordingto various embodiments from application to application. The effectiveintegration time of a given analyte band can correspond to a time wherethe portion of the wavelengths of the light signals in the analyte bandbeing integrated moves across two pixels on the detector array, or tothe entire time the portion of the wavelengths of the light signals inthe analyte band being integrated is in the detection zone, or to anytime therebetween. In addition, the portion of the wavelengths of thelight signals in the analyte band being integrated can, according tovarious embodiments, include (1) a range of wavelengths about a peakintensity of the light signals extending over two pixels of the detectorarray; or (2) a range of wavelengths including all wavelengths of thelight signals, or (3) a range of wavelengths anywhere between (1) and(2) above. By way of example, the integration time can include a time itwould take for the detector to integrate a range of wavelengths of theanalyte band corresponding to a full width of the intensity curve of thelight signals in analyte band at half of the peak or maximum intensityof the intensity wire, or to its “full width at half max.” The portionof the wavelengths of the light signals in the analyte band beingintegrated can depend on the number of different colors beingintegrated, and on how well the colors are separated from one another inthe emission spectra. As a general rule, the better separated the colorsin the emission spectra, the wider the portion of the wavelengths of thelight signals, and, hence, the longer the effective integration time. Inthe context of various embodiments, the “effective integration time ofan analyte band” therefore, as defined above, corresponds to theintegration time for a portion of the wavelengths of the light signalsin the analyte band, where the portion can include all of thewavelengths or a range thereof.

According to various embodiments, the use of TDI in collecting datapoints among other things addresses the problem of lowered irradiancewhen using irradiation sources emitting non-coherent light, such asLED's. The irradiance, that is, photons emitted per millimeters squared,is typically about a thousand times lower in LED's when compared withthe irradiance of lasers. TDI, according to the present invention, amongother things addresses the problem of lowered irradiance by allowing alonger period of time for the integration of signals from excitedmarkers. Related to TDI is the use of a broad detection zone accordingto the present invention. In a non-TDI detection system, the detectionzone is typically about one tenth of a millimeter squared. When usingTDI according to various embodiments, the detection zone for one channelcan be one hundred times larger, that is, about one millimeter squared,allowing a relatively larger number of markers to be excited and alarger number of data points to be integrated into a detector. The aboveprinciple of various embodiments can be equally applicable in instanceswhere a plurality of channels are used, the detection zones of each ofthe respective channels being adapted to be irradiated by at least oneirradiation source emitting non-coherent light.

For the purpose of accumulating charges to effect TDI, instead ofshifting the charges on the CCD as a function of the migration of theanalyte bands, various embodiments encompass within its scope moving theCCD itself and/or the image itself, that is, the light signals from thedetection zone, as a function of migration of the analyte bands suchthat the result of such movement is the tracking of each analyte band bya continuously moving row of accumulating photogenerated charge on theCCD during the effective integration time of the analyte band. By way ofexample, to accomplish the desired result mentioned above, appropriatemotors, gearing, belt drives, control units and power supplies can beused. For example, a linear actuator can be used to translate there-imaging optical system 30 and/or the CCD itself to minimize blurring.The image can in this way be made to be stationary on the CCD throughoutthe integration time. In such a case, a frame transfer CCD can be used.A frame transfer CCD has a parallel register that can be composed of twoCCD's arranged in tandem. The CCD register adjacent to the serialregister, or the storage array, is covered with an opaque mask andprovides temporary storage for charges during readout. The other CCDregister, or the image array, identical in capacity to the storagearray, is used for imaging. After the image array is exposed to light,the resulting charges are rapidly shifted in the parallel register up tothe storage array for subsequent readout. This shift operation typicallytakes a millisecond. While the masked storage array is being read, theimage array may integrate charge from the next image. See Charge-CoupledDevices for Quantitative Electronic Imaging, Photometrics Ltd. (1992),the content of which is incorporated herein in its entirety byreference.

Referring now to FIGS. 4, 5 a and 5 b, according to various embodiments,instead of one irradiation source, a plurality of irradiation sourcescan be provided to excite marker compounds in a sample. In theembodiment shown in FIGS. 4-5 b, the irradiation sources comprise fourLED's 50, 52, 54 and 56, the LED's being positioned so as to irradiatechannel-defining member 58, which defines two channels in the form oftwo capillary tubes as shown in particular in FIG. 5 b. Each LED emitsnon-coherent light in a predetermined range of wavelengths. For example,LED 50 and LED 52 emit substantially blue light, LED 54 emitssubstantially green light, and LED 56 emits substantially yellow light.As the above example shows, multiple LED's can be used to increase theavailable light. For example, if LED's 50 and 52 emit blue light, theycan be used simultaneously, either continuously or in a pulsed fashion,in this way increasing the amount of available blue light to obtain aproportional response in the associated markers. Although each type ofmarker used may ideally be excited by a different wavelength, LED's ofthe optimum wavelength and power level may not be available for eachgiven application. Hence, different markers may use the same LED.

The modulating optics according to the above embodiment of the presentinvention shown in FIGS. 4, 5 a and 5 b are comparable to the modulatingoptics in the embodiment shown in FIG. 1, with like components havingbeen labeled with like reference numerals. Thus, for each irradiationsource, a conditioning filter 18 and a optical component for focusing 19are provided, it being understood that the respective conditioningfilters and focusing lenses for the respective irradiation sources arenot, however, necessarily identical merely by virtue of the fact thatthey have been labeled with like reference numerals. As previously notedwith respect to FIG. 1, the function of each conditioning filter 18 isto let through only light in the wavelength range of excitation lightfor one or more of the markers. The conditioning filters 18 eachsubstantially block predetermined ranges of wavelengths of light emittedby the corresponding LED. The predetermined ranges correspond towavelengths of light that can overlap with the emission spectra of themarkers being excited by the corresponding LED. The function of eachoptical system 19 for focusing is to focus the conditioned light fromthe conditioning filter onto the detection zone 14, which, in theembodiment of FIGS. 4, 5 a and 5 b, corresponds to a respectivedetection zone for each of the shown capillary tubes. Excited markers indetection zone 14 thereafter emit light signals in the form of emittedlight 22. The light from the detection zone, as in the case of thefirst-mentioned embodiment of the present invention described inrelation to FIG. 1, includes the emitted light 22, and, in addition, aportion of the conditioned light 20 passing through the detection zone.

According to various embodiments, the light from the detection zone,labeled 20 and 22 in FIG. 5 a, can be collimated by collimating opticalsystem 24. The light thus collimated is thereafter passed through acorresponding bandpass filter 50′ on filter wheel 60 as shown in brokenlines in FIGS. 5 a and 5 b. It is noted that the filters in FIGS. 5 aand 5 b have been shown in phantom (broken lines) because, in thosefigures, the depiction of the filter wheel 60 is not cross-sectional,but rather represents plan views thereof.

Referring to FIG. 4, the filter wheel is shown in more detail assupporting therein a plurality of bandpass filters 51, 53, 55, and 57.Each of the bandpass filters is adapted to let through, substantiallyexclusively, predetermined wavelengths of light from the detection zonecorresponding to a portion of the wavelengths of the light signalsemitted by an associated marker. According to various embodiments, thereis a bandpass filter provided for each associated marker. The portion ofthe wavelengths of the light signals can include all of the lightsignals, or it can include, for each marker, a range of wavelengthsabout the peak intensity of light signals. For example, the range ofwavelengths about a peak intensity of emitted light signals can bebetween about 5% to about 20% of wavelengths on each side of the peakfor a given marker, or it can include the range of wavelengths at abouthalf of the intensity of the peak, generally called “full width at halfmax.” Thus, by way of example, bandpass filter 51 is adapted to filtertherethrough light signals emitted by given markers responsive to LED50. In FIGS. 5 a and 5 b, the apparatus according to various embodimentsis depicted in a mode where LED 50 irradiates the detection zone 14.However, it is clear that any of the shown LED's can be selectively usedto irradiate the detection zone 14. The filter wheel 60 (FIG. 5 a) canbe actuated by a filter wheel mechanism 62 (FIG. 5 a) that controls thefilter wheel to selectively position, in the path of the collimatedlight, the bandpass filter corresponding to the marker excited by theactive LED, that is, by the LED being used to irradiate the detectionzone. Filter wheel mechanism 62 may be controlled by a microprocessor orother similar device (not shown) as would be recognized by one skilledin the art. Similar to the embodiment in FIG. 1, the thus filtered lightis focused by a re-imaging optical system 30 onto an array 34 ofsolid-state detectors or detector array 34 such as onto thephoto-detecting surface of the parallel register of a charge-coupleddevice such as CCD 32. It is to be noted that various embodiments caninclude within its scope the provision of a single LED to excite allmarkers, the provision of multiple LED's to excite given ones of themarkers and the use of one LED per marker, the selection and number ofbandpass filters being a function of the markers themselves.

As seen more clearly in FIG. 4, the filter wheel according to variousembodiments can further include a filter thereon adapted to let throughonly light signals from the capillaries generated by a fifth marker.Four markers can be used to label the moving analytes where the analytesare DNA fragments, each marker corresponding to a given one of the basesin a DNA chain, that is, to the purines A (adenine) and G (guanine) andto the pyrimidines C (cytosine) and T (thymine). In addition, accordingto various embodiments, a fifth marker can, for example, be used forfragment analysis of the analytes. The fifth marker may be any marker,such as a dye marker, for doing fragment analysis. It is to be notedthat, according to various embodiments, the number of markers that canbe used are not limited to four or five as stated in the above example,but are rather limited only by the number of dyes available on themarket and responsive to the irradiation source or sources being used,based on application needs. Fragment analysis can be accomplished using,for example, the GENESCAN® Analysis software produced by AppliedBiosystems, Inc., Foster City, Calif. The GENESCAN® Analysis softwarecalculates the size of the unknown analytes by generating a calibrationor sizing curve based upon the migration times of the analytes in astandard that have been labeled with a marker. The unknown analytes aremapped onto the curve and converted from migration times to sizes. Inthe case of the embodiment shown in FIG. 4, the fifth marker filter FDon filter wheel 60 lets through light signals corresponding to markersused to label analytes in the standard. These markers are excitable byat least one of the irradiation sources 50, 52, 54, and 56 appropriatelymounted to allow fragment analysis.

Referring now to FIGS. 6 a through 6 e, these figures depict imagesproduced by moving analyte bands on the detector array 34 of CCD 32shown in the embodiment of FIGS. 4, 5 a and 5 b. Images are shown foreach of the markers used to label the analytes and which are responsiveto excitation by a given one of the irradiation sources 50, 52, 54, and56. Each shown frame of photo-detecting surface 36 in FIGS. 6 a through6 e shows two lanes of analyte bands each corresponding to one of thetwo capillaries of channel-defining member 58. The bands move along thedirection of migration M, and are shown as being limited on each sidethereof in the spectral direction by virtue of the light from themarkers having been filtered through a corresponding bandpass filter. Atthe right of each frame is shown an intensity profile 45 correspondingto the capillary for which the charges are produced on photo-detectingsurface 36 on the right lane thereof.

According to various embodiments, the intensity profiles are, accordingto known methods, aligned and combined, and thereafter can bemulticomponented in order to account for any spectral overlap. Similarto the embodiment of FIG. 1, the serial register of the CCD 32 in theembodiment of FIGS. 4, 5 a, and 5 b collects the charges accumulated foreach analyte band during its integration time. All signals received fromthe detector can be converted from analog to digital and conveyed to aserial port for transmission to a multipurpose computer for storage andfor further processing and analysis. Alternatively or additionally, theanalog output could be sent directly to an output device for display orprinting. By way of example, a multipurpose computer may be used toperform the multicomponenting process. Multicomponenting is a processthat is known to a person skilled in the art, and that can involve aspectral calibration within a multicomponenting software program. Thespectral calibration can be obtained through a predetermined signaturematrix corresponding to each marker. Each signature matrix provides asignature snapshot of the intensity of light signals by a given markeras a function of the wavelengths of those light signals. By virtue ofthe signature matrices, a combination of intensity curves for a givenwavelength band emitted from the detection zone can be broken down intoits components corresponding to light signals emitted by individual onesof the markers. In this way, a relatively accurate assessment of thelight signals by respective ones of the markers can be made for thedetection process.

In operation, the apparatus according to the embodiment of the presentinvention shown in FIGS. 4, 5 a, and 5 b effects an irradiation ofdetection zone 14 by each respective one of the irradiation sources, insequence. The filter wheel is in each case adjusted to dispose beforecollimating optical system 24 a bandpass filter 51, 53, 55, 57, or FDcorresponding to the marker being used. The detection zone can be,according to various embodiments and found in the embodiments shown inFIGS. 4, 5 a and 5 b, irradiated for the duration of the integrationtime of the moving analyte bands across the detection zone. During theintegration time, similar to the embodiment described in relation toFIG. 1, the charges generated by the light signals for markers beingdetected can be moved along the parallel register in the direction ofmigration and are accumulated in the detector or CCD 32 before they areread. Thereafter, another respective one of the irradiation sources isused to irradiate the detection zone to excite corresponding ones of themarkers, while a corresponding bandpass filter is positioned before thecollimating optical system 24 by filter wheel 60. Again, light signalsfrom the excited markers are detected by detector 32, the chargesassociated therewith being accumulated in the detector during theintegration time before the accumulated charges are read. The aboveprocess can continue until all of the irradiation sources haveirradiated the detection zone, and until all filters, including filterFD, have been positioned before the collimating lens to filter the lighttherefrom. Detector 32 can be a frame transfer CCD, such that each frameof the CCD upon which charges have been accumulated can be transferredto a storage array, the image array therefore being available for thenext series of charge accumulations produced by the next respective oneof the irradiation sources being used.

The above process may be repeated in cycles as many times as necessaryin order to obtain sufficient data regarding each analyte beingdetected. Fewer cycles typically result in an increase in signal. Thisis because fewer cycles mean longer integration times, and thereforeincreased readout signals over the noise typically associated with aCCD. On the other hand, increasing the number of cycles can improve thedynamic range of the system. The dynamic range of the system is definedas the largest peak signal that can be read by a given CCD (or “fullwell capacity”) over the smallest peak that can be read by the CCD justabove the noise level. A CCD typically has a given full well capacity.If a peak signal is above the full well capacity of a CCD, it will beoff the scale of the CCD. Short integration times allow peak signals tobe generally attenuated so as to reduce the possibility of saturatingthe CCD with off-scale signals, that is, with signals that go beyond theCCD's full well capacity. In this way, analyte concentrations may beincreased while still allowing the CCD to reliably detect signal levelswithout saturation. According to various embodiments, there is atrade-off between the use of fewer cycles amounting to longerintegration times (such as, for example, 5 seconds) and the use of morecycles amounting to shorter integration times (such as, for example, 1second). Longer integration times are useful where the noise level isrelatively high and where the sensitivity of the system needs to beincreased in light of the same. On the other hand, where the noise levelin the system is relatively low, multiple reads may be taken of signalsfrom the same marker, and the read signals may thereafter bemulticomponented, the CCD in this way allowing the detection of brighterpeaks without going off-scale.

By way of example, a frame transfer CCD may be controlled to collect thelight signals corresponding to the blue marker during integration time twhile the LED exciting primarily the blue marker irradiates thedetection zone. Thereafter, the entire CCD is read out. The filter wheelis then switched to the bandpass filter associated with the greenmarker, and the LED exciting primarily the green marker irradiates thedetection zone. The CCD then collects the light signals corresponding tothe green marker during integration time t. The entire CCD is then readout. The filter wheel is then switched to the bandpass filter associatedwith the yellow marker, and the LED exciting primarily the yellow markerirradiates the detection zone. The CCD then collects the light signalscorresponding to the yellow marker during integration time t, and theentire CCD is thereafter read out. The above process can then berepeated for all five markers in this example and as described inrelation to FIGS. 4, 5 a and 5 b. As suggested above, in this example,the band takes about five times the integration time from the top of theframe transfer CCD to the bottom thereof, that is, to the readoutregister. Each readout of the CCD corresponds to one marker. All of thereadouts can then be aligned and combined in a known manner formulticomponenting.

An example of the manner in which multicomponenting may be effected isshown in FIGS. 6 f through 6 h. Here, it is assumed that filters 51, 53,55, 57, and FD let through wavelengths of light in the blue, green,yellow, red and “fifth” portions of the spectrum. The “fifth” portionmay, for example, be in the orange range of the spectrum. FIG. 6 f is aschematic representation of an electropherogram showing fluorescenceintensity curves for each filter of the filter wheel in FIG. 4 duringthree readings of the signals by detector 32. The intensity curvescorrespond to a reading of light signals from an analyte labeled with amarker such as FAM, that is, a dye marker that emits light signalsmostly in blue. The first portion of each curve, drawn in solid lines,corresponds to a reading from each one of the blue filter, green filter,yellow filter, red filter and fifth filter of FIG. 4 during a firstintegration time t for each filter. The second portion of each curve,drawn in broken lines, corresponds to a reading from each of thementioned filters during a second integration time t. The third portionof each curve, drawn in solid lines, corresponds to readings from eachof the mentioned filter during a third integration time t. In theschematic representation of the electropherogram of FIG. 6 f, thehorizontal axis corresponds to distance traveled by the analyte, and thevertical axis corresponds, for each filter, to the fluorescenceintensity of light emerging from that filter. Thus, the first set ofcurves in solid lines represents intensity curves for light emergingfrom each filter during a first cycle of the filter wheel 60. The secondset of curves in broken lines represents intensity curves for lightemerging from each filter during a second cycle of the filter wheel 60.The third set of curves in solid lines represents intensity curves forlight emerging from each filter during a third cycle of the filter wheel60. As seen in FIG. 6 f, the light from the blue filter exhibits themost intensity during each cycle, the intensity decreasing as light iscollected from the green filter, the yellow filter, the red filter andthe fifth filter, respectively. As seen in the instant example,therefore, spectral overlap causes FAM to emit mostly in blue, some ingreen, less in yellow, etc. As, within each cycle, filter wheel 60 isrotated to place a subsequent filter in the path of fluorescence fromthe detection zone 14, the analyte has moved a distance x as marked onFIG. 6 f after an integration time t.

Referring now to FIG. 6 g, that figure is a schematic representation ofthe intensity curves of FIG. 6 f in aligned format formulticomponenting. Here, each intensity curve other than the onecorresponding to blue light is shifted by a multiple of x as a functionof the filter to be aligned with the intensity curve corresponding toblue light. After being aligned, the intensity curves are combined andmulticomponented, thus yielding the intensity curve for FAM shown inFIG. 6 h. To the extent that only FAM is being detected in the exampleof FIGS. 6 f through 6 h, the intensity curves for JOE, TAMRA, ROX, andthe fifth dye are all flat in FIG. 6 h, as recognizable by one skilledin the art.

Referring now to FIGS. 6 i and 6 j, representative excitation efficiencycurves and fluorescence intensity curves are shown as having beenplotted versus wavelength for four different dye markers that can beused in oligosynthesis, namely, 5-FAM, JOE, TAMRA, and ROX. These dyemarkers are exemplary of those that can be used in various embodimentswhere a plurality of dye markers are to be used, such as the embodimentsshown in FIGS. 4, 5 a, 5 b, and 7.

As seen in FIG. 6 i, the x-axis corresponds to wavelengths, expressed innanometers or nm, emitted by an irradiation source, and the y-axiscorresponds to the percentage of excitation efficiency. Here, it can beeasily appreciated that 5-FAM has its maximum absorbance, correspondingto its peak percent excitation efficiency, at about 490 nm. The maximumabsorbance at a given wavelength indicates that the dye marker beingconsidered fluoresces at its peak fluorescence intensity when it isirradiated at the given wavelength. As further seen in FIG. 6 i, JOE hasa maximum absorbance at about 526 nm, TAMRA has a maximum absorbance atabout 560 nm, and ROX has a maximum absorbance at 588 nm. Thewavelengths on the x-axis could be emitted by any irradiation source,such as, for example, an LED. FIG. 6 i also shows that, where a dyemarker, such as 5-FAM, is being irradiated at its maximum absorbancewavelength, other dye markers, such as, for example, JOE, TAMRA, andROX, do exhibit some absorbance, although to a lesser extent whencompared to 5-FAM.

Referring now to FIG. 6 j, the x-axis corresponds to the wavelengths offluorescent light, expressed in nm, emitted by excited dye markers. They-axis corresponds to the percentage of fluorescence intensity. As seenin FIG. 6 j, 5-FAM has a peak fluorescence intensity at about 522 nm,JOE has a peak fluorescence intensity at about 554 nanometers, TAMRA hasa peak fluorescence intensity at about 582 nm, and ROX has a peakfluorescence at about 608 nm. The wavelengths on the x-axis are emittedby the four mentioned dye markers. FIG. 6 j also shows that, where a dyemarker, such as TAMRA, fluoresces at its peak fluorescence intensity,other dye makers, such as 5-FAM, JOE, and ROX, also fluoresce, althoughat lesser fluorescence intensities.

When light is emitted from the dye markers in the detection zone atvarious colors, each dye marker can be excited efficiently, and furtherin a way that will allow its detection by way of its unique spectralsignature. When two dye markers exhibit fluorescence intensity peaksthat are close together, that is, for example, when the differencebetween the fluorescence intensity peaks of two dye markers is less thanabout 30 nm, there is a high level of overlap of the light emitted bythose two dye markers. A high level of overlap makes it harder todistinguish between the light emitted by the two dye markers, andtherefore makes it harder to determine the relative amounts of the twodye markers. Typically, there is usually overlap present in the lightemitted by different dye markers, as suggested for example in FIGS. 6 iand 6 j. However, the overlap can be minimized by selecting dye markersthat present easily distinguishable fluorescence intensity peaks, suchas those shown in FIG. 6 j. According to various embodiments, it ispossible to first start with an irradiation source emitting radiationwithin a given range of wavelengths, and to investigate each suchirradiation source to see how well it excites the dye markers available.Graphs such as those shown in FIGS. 6 i and 6 j could be used in thiscontext. For example, where excitation efficiency curves of various dyemarkers are plotted in the manner shown in FIG. 6 i, an irradiationsource, such as an LED emitting light in a given range of wavelengths,can be predicted to excite given ones of the dye markers based on theirexcitation efficiencies. Thereafter, from a fluorescence intensity graphsuch as in FIG. 6 j, it becomes possible to determine how much overlapwould exist between intensity peaks of the different markers. From sucha determination, it then becomes possible to choose which set of markerswould be best suited for a particular application. Once the dye markershave been chosen, it then becomes possible to choose the filterscorresponding thereto, such as the filters shown on the filter wheel ofthe embodiment FIG. 4 described above, and on the filter wheel in theembodiment of FIG. 7 described below. The best set of markers wouldexhibit the desired minimum amount of overlap at the emitted wavelengthsthat are to be detected.

Because each marker has a different excitation curve, the fluorescenceoutput can be dramatically increased by the use of an LED that is wellmatched to this marker. This provides an increase in the desired light,that is, in the emission from the marker of interest, while minimizingundesired light, that is, background light from the system and emissionsfrom other markers. This results in data that is of better quality. Forexample, if a blue/green LED with an excitation maximum of 503 nm isused for the marker designated FAM, the absorption will be high for FAM,at about 80%, and low for the marker designated ROX, at about 6%.Similarly, a yellow LED with an excitation maximum of 592 nm will notexcite FAM, while its absorption in connection with ROX will be about90%.

Referring now to FIG. 7, another embodiment is depicted. This embodimentis similar to the one shown in FIGS. 4, 5 a, and 5 b to the extent thata number of irradiation sources are used to sequentially irradiate thedetection zone. However, the embodiment of FIG. 7 differs from theembodiment of FIGS. 4, 5 a, and 5 b in a number of respects. The premisebehind the embodiment of FIG. 7 is to allow a reading of light signalshaving wavelengths in differing frequency ranges on the same array ofthe detector, the charges corresponding to the generated light signalsbeing spatially offset as a function of the bandpass filter being usedin connection with the markers emitting those light signals.Advantageously, such an embodiment allows a continuous reading of theaccumulated charges on the detector array during time delay integrationor TDI, rather than a frame-by-frame reading as in the case of theembodiment of the present invention described above with reference toFIGS. 4, 5 a, and 5 b.

In the embodiment of FIG. 7, the irradiation sources, together withassociated optics such as the conditioning filter 18 and the opticalsystem for focusing 19, can be provided on an irradiation source wheel61 as shown. As seen in FIG. 7, components of the apparatus that aresimilar to those in the embodiment of FIG. 1 have been labeled with thesame reference numerals, such as conditioning filter 18, focusingoptical system 19, collimating optical component or system 24, andre-imaging optical component or system 30. The irradiation wheel 61 isrotatable to selectively position each respective one of the irradiationsources and associated optics to irradiate detection zone 14. Fourirradiation sources similar to sources 50, 52, 54, and 56 in FIGS. 4, 5a, and 5 b, can be provided. In addition, a filter wheel 61′ can beprovided, similar to filter wheel 60 in FIGS. 4, 5 a, and 5 b. Therotation of both wheels 61 and 61′ can be effected by the provision of afilter wheel drive 62 similar to the filter wheel drive of theembodiment of FIGS. 4, 5 a, and 5 b described above. According tovarious embodiments, the two wheels 61 and 61′ can further be coupled toone another and to the filter wheel drive 62 by way of a rotatable shaft63 as shown. It is noted that various embodiments encompass within itsscope the provision of a plurality of irradiation sources that are notnecessarily provided on an irradiation wheel, or the provision of anarrangement where the two wheels 61 and 61′ are not coupled to oneanother by a shaft, but are rather actuated independently by their ownrespective wheel drives.

In the embodiment of FIG. 7, the apparatus is provided with an offsetsystem 64, which may be disposed either on the filter wheel 61′ inassociation with a corresponding bandpass filter, or coupled to at leastone of the detector 32, the modulating optics, and the detection zone14, for spatially offsetting the light signals impinging upon the arrayof the detector by a predetermined amount as a function of the bandpassfilter being used. In effect, the offset system is, according to variousembodiments, adapted to offset the light signals impinging upon thearray 34 of detector (CCD) 32 by a predetermined amount for eachbandpass filter. The offsetting may be accomplished by providing anoffset system 64 that includes a plurality of offset mechanisms 66,shown in broken lines in FIG. 7, and disposed on filter wheel 61′. Eachoffset mechanism is associated with a respective one of the bandpassfilters to offset the filtered light therefrom. In such a case, theoffset mechanisms 66, one of which is shown in FIG. 7, may comprisegratings, mirrors, prisms, or any other devices for offsetting light, asreadily recognizable by those skilled in the art. Mechanisms 66 can bedistributed about the circumference of filter wheel 61′ in front of eachcorresponding bandpass filter, with wheel 61′ in FIG. 7 being otherwiseidentical to wheel 60 in FIG. 4. In the alternative, the offsetting canbe accomplished by providing an offset control device 67, also shown inbroken lines, and can be coupled to at least one of the detection zone14, the modulating optics, and the detector array 34, for offsetting thelight signals impinging upon the array 34 of the detector 32. By way ofexample, the offset system may move at least one of the detection zone14, collimating optical system 24, re-imaging optical system 30, ordetector array 34 by a predetermined amount in order to thereby offsetthe light signals impinging upon the detector array 34. In this latteralternative of the offset system, the system can include any suitabledevice for effecting a translational movement of at least one of thedetection zone 14, collimating optical system 24, re-imaging opticalsystem 30, or detector array 34, as would be recognized by those skilledin the art. Such devices can include, for example, solenoids, or motordriven linear actuators such as lead screws, rack-and pinion systems,cams, and the like. For example, a cam can be attached to drive shaft 63to cause a translation of the re-imaging optical system 30 in a numberof ways recognizable by one skilled in the art. The predetermined amountcan correspond to a one to one ratio relative to the amount by which thelight signals impinging upon the detector array are sought to be offset.The range of wavelengths corresponding to the predetermined amount is afunction of a number of different factors, such as, for example, theirradiation source being used and the markers being excited. However,once a given set including an irradiation source and its correspondingbandpass filter have been positioned to irradiate the detection zone andto filter light therefrom, the predetermined amount by which the lightsignals are to be offset may be readily determined by determining whereon the detector array the charges produced by those light signals shouldbe situated with respect to the detector array itself, and with respectto light signals in different wavelength frequency ranges. Thus, whereindividual mechanisms 66 are used in conjunction with a correspondingbandpass filter to offset the light emerging therefrom, each mechanism66 is chosen according to the frequency ranges of wavelengths that thebandpass filter lets through. In the alternative, where offset controldevice 67 is used, the offset control may be programmed to offset atleast one of the detection zone 14, the modulating optics, and thedetector array 34, with respect to one another by the predeterminedamount. The offsetting could, by way of example, be accomplished bymoving either the detection zone 14, the modulating optics, or thedetector array 34, in a translational motion by the predeterminedamount, the image created by the light signals being spatially offsetcorrespondingly. The embodiment shown in FIG. 7 can involve the use of aplurality of LED's similar to those used in the embodiment of FIGS. 4, 5a, and 5 b. The offset amount should be sufficient be prevent overlap ofthe images from each bandpass filter.

It is noted that offset system 64, including offset mechanisms 66, or,in the alternative, offset control device 67, are shown in broken linesin FIG. 7 in order to suggest that mechanisms 66 and offset controldevice may be used as alternatives of the offset system 64. It isfurther noted that various embodiments encompass within its scopeinstances where both alternatives, that is, mechanisms 66 and offsetcontrol device 67, are used in conjunction with one another. Moreover,the modulating optics, according to various embodiments, comprise atleast one of conditioning filter 18, focusing optical system 19,collimating optical system 24, and re-imaging optical system 30,encompasses any devices or system for achieving the functions associatedwith the components listed above as would be within the knowledge ofpersons skilled in the art. In addition, with respect to offset controldevice 67, where the instant disclosure describes a coupling of device67 to the modulating optics, what is meant is that the offset controldevice 67 is coupled to at least one of the components of the modulatingoptics. It is further to be noted that, although the embodiments ofFIGS. 1 and 4-5 b depict a set of two capillaries being analyzed,various embodiments encompass a detection zone defined by any suitablechannel-defining member, such as any number of capillaries, any numberof channels in an etched plate, and even a slab plate. According tovarious embodiments, the channel-defining member can assume anyorientation according to application needs, such as a horizontalorientation or a vertical orientation. Moreover, the embodiments ofFIGS. 4, 5 a, and 5 b are not necessarily limited to four irradiationsources and five bandpass filters, but encompass the use of any suitablenumber of bandpass filters depending on the markers being used. It isfurther to be noted that, in the described embodiments, any number ofdifferent irradiation sources, such as LED's, emitting light in anynumber of wavelength ranges, may be used. Limitations in this regard mayonly be a function of the available space, the availability of LED's,and/or the types of markers that can be used. Additionally, whenever an“optical system” is referred to, this system may be a single lens, alens system, a mirror system, or any other optical system capable offulfilling the desired and stated functions, as readily recognizable bythose skilled in the art.

In operation, the detection zone 14 in FIG. 7 is irradiated by a firstone of the irradiation sources, such as, by LED 50 as depicted in FIG.7. The light signals emitted by the markers excitable by the light fromLED 50 are, as previously described, filtered through a correspondingbandpass filter 51, and thereafter focused onto detector array 34 by are-imaging optical system. Where offset system 64 includes mechanisms66, the filtered light from bandpass filter 51 is offset by mechanism 66by the predetermined amount corresponding to the range of wavelengthsthat the bandpass filter lets through, as described above. Eachsubsequent irradiation source and corresponding bandpass filter are thenpositioned to irradiate the detection zone and to filter the lighttherefrom through a rotation of the wheels 61 and 61′ in conjunctionwith one another. As previously suggested, any number of markers,bandpass filters and LED's may be used in the shown system.

In the embodiment of FIG. 7, charges are accumulated for each respectiveirradiation source during the integration time of analyte bandsexcitable by light from the respective irradiation source. Theaccumulation of charges may be effected, as previously described, eitherby shifting the charges on the detector array, and/or by moving,relative to one another, the detector array and light signals from thedetection zone. After each integration time, the wheels 61 and 61′ arerotated to position the next set, including an irradiation source and abandpass filter, in a functional position, that is, in a position forthe irradiation source to irradiate the detection zone and for thebandpass filter to filter the light from the detection zone. The aboveis carried out until one cycle is completed, that is, until all of theirradiation sources and filters have been used. The above cycle may berepeated as many times as necessary on an application-by-applicationbasis. To the extent that the embodiment of FIG. 7 includes an offsetsystem for spatially offsetting light signals in differing frequencyranges, the embodiment allows a continuous reading of accumulatedcharges by the detector, thereby making possible a continuous time delayintegration of the light signals from the analytes in a sample into thedetector array 34. Unlike the embodiment of FIGS. 4, 5 a, and 5 b, wherethe accumulated charges corresponding to each range of wavelengths oflight signals are read and discarded before charges for the next rangeof wavelengths are read, the embodiment of FIG. 7 allows the accumulatedcharges from each wavelength range to be read in a continuous fashion.

Referring now to FIG. 8, the image produced by the moving analyte bandson the array of detector (CCD) 32 is shown for each of the wavelengthranges and each of the two capillaries 58. In the shown image, eachrange of wavelengths is assigned an arbitrary color, the ones showntherefore being arbitrarily referred to as “blue,” “green,” “yellow,”“red,” and “fifth.” For each color, the column on the left correspondsto charges produced by light signals from one capillary, and the columnon the right to charges produced by the next capillary. As shown, thearray features charges generated by light signals across the color axisλ, offset with respect to one another by offset system 64 in the mannerpreviously described. Although in the shown embodiment, five arbitrarycolors are given by way of example, the number of wavelength ranges usedwill be dependent on the particular application, and can range from oneto as many as the system supports. It is noted that the λ axis isreferred to here as the “color axis” rather than the “spectral axis,”because there is no need for the colors, one for each bandpass filter,to be arranged from shorter to longer wavelengths. According to theembodiment as shown in FIG. 7, to the extent that charges from lightsignals of differing wavelengths may be produced on the same array oflight signals so as to be spatially offset with respect to one another,as seen in FIG. 8, those charges may be accumulated during theintegration time and read by the detector on a continuous basis. Thiseliminates the need to take the time to shut off the detector in orderto allow a reading of charges in a given range of wavelengths on aframe-by-frame basis, and/or eliminates the need for a frame transferCCD. In addition, the above embodiment facilitates a longer integrationtime, simpler data output and a less complex CCD.

According to various embodiments, the detection zone, including one ormore channels, or a slab gel, may be irradiated with multiple-colorirradiation sources, such as multiple-color LED's. As in the embodimentsof FIGS. 1 and 4-5 b described above, the use of multiple-color LED'scan greatly improve the absorption efficiency of some of the markers.The potential problem that would need to be overcome with such anarrangement would be the elimination of excitation light. Long passfilters or wide band pass filters are conventionally used to reject theexcitation light, but only with irradiation sources such as lasersources. According to various embodiments, the irradiation source isconditioned to provide only a narrow wavelength range of light and abandpass filter is used to substantially block all of the unwantedexcitation light. An example of the results, in the form of graphs, ofimplementing such an embodiment of the present invention is provided inFIGS. 9 a through 9 d.

As depicted in FIGS. 9 a through 9 d, a detection zone of an apparatushas been irradiated by two irradiation sources simultaneously, althoughany number of irradiation sources is possible according to variousembodiments. In such a case, a set-up of the system may be used such asthe one shown in FIG. 1, except that the single LED in FIG. 1 isreplaced with a set of double LED's, the associated optics being alteredaccordingly. In particular, for each set of double LED's, a conditioningfilter is used. Here, the conditioning filter has the role ofsubstantially blocking predetermined ranges of wavelengths of lightemitted by the LED as previously described, each predetermined rangecorresponding to each LED, as suggested, for example, in FIG. 9 a. FIG.9 a shows a graph of relative excitation intensity for each LED, versuswavelength expressed in nm. The graph of FIG. 9 a suggests that twoLED's are being used, respectively, generally in the violet and orangeportions of the spectrum. However, it is to be understood that variousembodiments are not limited to the above two types of LED's, butencompasses any number of LED's emitting light in any range offrequencies according to application needs. As seen in FIG. 9 b, eachpredetermined range that passes through the conditioning filtercorresponds to wavelengths emitted by each example of the LED depictedin the graphs, and capable of exciting the markers responsive to eachLED. FIG. 9 b is a graph of percent transmission of light through theconditioning filter versus wavelength. The behavior of the conditioningfilter corresponding to the graphs of FIGS. 9 a-9 d substantially blocksall excitation light except for sections around the LED emissionmaximum. Wavelength ranges correspond to a range of from about 450 nm toabout 490 nm, and to another wavelength range of from about 580 nm toabout 605 nm, as depicted in FIG. 9 b. Again, it is noted that theconditioning filter, and the predetermined ranges passing therethrough,are, among other things, functions of the LED set being used, and, tothe extent that any number of LED's may be used emitting light in anyranges of frequencies, the conditioning filter is chosen accordingly.

It is further to be noted that the conditioning filter of variousembodiments may include a single conditioning filter, or a series ofconditioning filters capable of filtering the light from the LED's aspreviously described. The conditioned light is, as previously describedabove in relation to FIGS. 1 and 7, focused onto the detection zone ofan electrophoretic detection system. The light emitted by markers in thedetection zone is then passed through a bandpass filter. The function ofthe bandpass filter is to let through, substantially exclusively,predetermined wavelengths of light from the detection zone correspondingto a portion of the wavelengths of the light signals emitted by anassociated set of markers, to thereby produce filtered light. Thebandpass filter's function is to let through only a portion of the lightemitted by an associated set of markers, and not the excitation light bythe LED's that passes the detection zone. The portion of the wavelengthsof the light signals can include all of the light signals, or it caninclude, for each marker, a range of wavelengths about the peakintensity of light signals. For example, the range of wavelengths aboutthe peak intensity of light signals can be between about 5% to about 20%of wavelengths on each side of the peak wavelength of a given marker, orit can include a range of wavelengths at about half of the intensity ofthe peak wavelength, or “full width at half max.” The collection of thepredetermined wavelengths can be performed using a dispersion approach,such as the approach shown in FIG. 1, or by additional bandpass filtersas shown in FIG. 4.

As seen in FIG. 9 c, where percent light transmission is plotted versuswavelength, the particular bandpass filter being used, the behavior ofwhich is shown in the figure, lets through light corresponding to the“blue,” “green,” “yellow,” “red,” and “orange” markers. The regions orzones corresponding to the excitation light by the LED's are blocked. Asnoted previously with respect to the conditioning filter, the bandpassfilter in various embodiments may include a single bandpass filter, or aseries of bandpass filters capable of filtering the light from the LED'sas previously described.

FIG. 9 d plots relative emission intensity versus wavelength, expressedin nm, for the light let through by the bandpass filter. FIG. 9 d, ineffect, provides a breakdown, by wavelength, of the light transmittedthrough the bandpass filter. As shown in FIG. 9 d, the markers beingexcited by the LED's used in the example of FIGS. 9 a through 9 d emitlight in the “blue,” “green,” “yellow,” “red,” and “orange” ranges ofwavelengths. Before focusing the light signals thus filtered onto thedetector array of a CCD, a dispersion element can be used, such asgrating 28 shown in FIG. 1. In such a case, the resulting image would besimilar to that in FIGS. 2 and 3, except that the image will have one ormore dark zones corresponding to the blocked excitation light. Theexistence of one or more dark zones, however, does not prevent theability to perform multicomponenting to determine the intrinsic dyeconcentrations of each band in an electropherogram. Accordingly, thisembodiment of the present invention that would generate graphs such asthose of FIGS. 9 a-9 d allows the use of multiple-color LED's. TDI canstill be performed in any of the previously described manners.

When irradiating multiple-channels with multiple-color LED's, accordingto various embodiments, the channels may be selectively masked in orderto keep the light signals generated by the markers in each channelseparate from one another. As seen by way of example in FIGS. 10 and 11,where three channels 68 a, 68 b, 68 c are provided, the channels areselectively masked using a mask 70 to create three windows 72 a, 72 b,and 72 c in the irradiation zone 67. The mask can be made of ametallized surface on a glass or fused silica plate that is disposedadjacent, for example, immediately above the channels. The metal can bedeposited in a controlled manner by photolithography methods, as readilyrecognizable by one skilled in the art of micro-machining to cover theentire plate except in the areas forming the windows. Alternatively,windows can be cut out of an optically non-transparent plate, or a platewith windows can otherwise be formed. As seen in FIG. 11, the array 34of the detector is configured such that it is divided into three frames74 a, 74 b, and 74 c, respectively, corresponding to windows 72 a, 72 b,and 72 c, for allowing TDI to be effected on the charges generated bylight signals detected through the respective windows on awindow-by-window basis.

The above concept is more fully illustrated in FIG. 12 a, where a frameis partially cut-away to show a schematic representation of the portionof detector array 34 corresponding to frame 74 a for detecting lightpassing through window 72 a. Here, each band 76 moves in the migrationdirection M, and the charge packets on the detector array 34 areaccumulated. The accumulation of the charges for effecting TDI may, asdescribed above, be brought about by a shifting of the charges on thedetector array in the migration direction M at a predetermined speedcorresponding to the average speed of migration of the analyte bands, assuggested in FIG. 12 a by the shifting movements depicted by referencenumeral 78. Data can be collected from frame 74 a from the top of theframe to the bottom of the frame. Some of the light is blocked by themask, causing a variable collection efficiency as suggested by FIG. 12b. The integration of the accumulated charges occurs in the embodimentof the present invention shown in FIGS. 10-12 b on a frame-by-framebasis as previously explained in relation to FIGS. 4, 5 a, and 5 b, theframe data being combined in a known manner to create anelectropherogram. It is further possible, according to variousembodiments, to abut the frames, that is, to eliminate any distancebetween them so as to combine the resulting images on detector array 34.According to various embodiments, the above arrangement can allow theseparation and detection of light signals from multiple-channels whilepermitting the simultaneous irradiation of those channels withmultiple-color irradiation sources. The above is made possible throughthe use of a single camera instead of one camera per channel, the singlecamera maintaining a spatial separation of light signals from eachmasked channel.

According to various embodiments, the modulating optics that can be usedin the electrophoresis arrangements are disclosed in U.S. applicationSer. No. 09/564,790, the content of which is incorporated herein in itsentirety by reference. In particular, in the above-referencedapplication, the modulating optics shown in FIG. 1, using the cat's eyeaperture of FIG. 24, can be useful in electrophoresis arrangementsaccording to the various embodiments.

Various embodiments can further pertain to an apparatus for detectinganalytes in a sample, and can comprise: means defining at least onechannel therein having a detection zone; means for separating a samplecontaining analytes and disposed in contact with a migration mediumdisposed within the at least one channel into analyte bands migratingalong the at least one channel, wherein each analyte band is detectableby the presence of a marker; means for irradiating the detection zonewith non-coherent radiation, that can thereby excite markers responsiveto the radiation and which emit light signals indicative ofcorresponding analytes; means for detecting the light signals bycollecting the light signals that can thereby produce chargescorresponding to the light signals; means for effecting a time delayintegration of the light signals within the detector array byaccumulating within the detector array the charges corresponding tolight signals associated with at least one given analyte band during anintegration time of the at least one given analyte band moving acrossthe detection zone; and means for reading the accumulated charges. Theabove-described means have been substantially shown and described inFIGS. 1 through 15.

It is to be understood that various embodiments are useful in detectingand imaging not only fluorescent labeled molecules, but also proteins,virus, bacteria, etc., which are electrophoretically or otherwiseseparated on a variety of carriers, such as in capillary tubes, andacross, on, in, or through slab gels, membranes, filter paper, petridishes, glass substrates, and the like.

Other embodiments of the present teachings will be apparent to thoseskilled in the art from consideration of the present specification andpractice of the present teachings disclosed herein. It is intended thatthe present specification and examples be considered as exemplary onlywith a true scope and spirit of the teachings being indicated by thefollowing claims and equivalents thereof.

1. An apparatus for detecting analytes in a sample, comprising: one ormore channels having a detection zone; one or more irradiation sourcesdisposed for irradiating the detection zone with radiation; a detectorarray disposed for collecting light signals emitted from markers in thedetection zone excited by the radiation, the detector array having anoutput; and a system coupled to the detector array for effecting timedelay integration of charges on the detector array corresponding to thelight signals by accumulating the charges before reading the charges atthe output of the detector array, the system effecting time delayintegration by moving, relative to one another, the detector array andthe detection zone such that the detector array tracks light signalsemitted from the markers.
 2. The apparatus according to claim 1, whereinthe system for effecting time delay integration accumulates the chargesby at least one of (a) shifting the charges on the detector array, and(b) moving, relative to one another, the detector array and lightsignals from the detection zone.
 3. The apparatus according to claim 1,wherein the one or more irradiation sources comprises one or more lightemitting diodes.
 4. The apparatus according to claim 1, furthercomprising modulating optics disposed between the detection zone and thedetector array, the modulating optics comprising a relay lens systemhaving a collimating lens and a re-imaging lens.
 5. The apparatusaccording to claim 4, wherein the modulating optics further include aconditioning filter disposed between the one or more irradiation sourcesand the detection zone.
 6. The apparatus according to claim 5, whereinthe modulating optics further include a focusing lens disposed betweenthe conditioning filter and the detection zone.
 7. The apparatusaccording to claim 4, wherein the modulating optics further include atransmission grating disposed between the focusing lens and there-imaging lens.
 8. The apparatus according to claim 4, wherein themodulating optics further include a filter disposed between thedetection zone and the detector array for filtering through only thelight signals.
 9. An apparatus for detecting analytes in a sample,comprising: one or more channels having a detection zone; one or moreirradiation sources disposed for irradiating the detection zone withradiation; a detector disposed for collecting light signals emitted frommarkers in the detection zone excited by the radiation, the detectorhaving an output; modulating optics disposed between the detection zoneand the detector; and a system coupled to the detector for effectingtime delay integration of the charges on the detector corresponding tothe light signals by accumulating the charges before reading the chargesat the output of the detector, the system effecting time delayintegration by moving one or more of the detection zone, the modulatingoptics, and the detector.
 10. The apparatus of claim 9, furthercomprising a re-imaging lens disposed between the detection zone and thedetector array for optically inverting an image produced by the lightsignals before the image is collected by the detector array.
 11. Anapparatus for detecting analytes, comprising: a plurality of elongatechannels; a light source; an excitation-light pathway extending fromsaid light source to said channels; focusing optics disposed along saidexcitation-light pathway; and a charge-coupled device optically coupledto said channels, wherein said charge-coupled device is configured tooperate in time-delay integration mode by moving the charge-coupleddevice to track an emission signal.
 12. The apparatus of claim 11,wherein said channels are defined by capillary tubes or grooved plates.13. The apparatus of claim 11, further comprising a separation mediumsupported by said channels.
 14. The apparatus of claim 11, including atleast four co-extensive channels.
 15. The apparatus of claim
 11. furthercomprising modulating optics disposed along an optical path extendingbetween said charge-coupled device and said channels.
 16. The apparatusof claim 11, wherein said light source is a light-emitting diode.
 17. Anapparatus for detecting analytes, comprising: a plurality of elongatechannels; a detection zone located at one or both of (i) along saidchannels and (ii) outside of said channels; a light source andassociated focusing optics configured for irradiating at least a portionof said detection zone with light; and a charge-coupled device opticallycoupled to said detection zone for collecting light signals emitted frommarkers in the detection zone excited by the light irradiated by thelight source; wherein said charge-coupled device is configured tooperate in time-delay integration mode, the apparatus effecting timedelay integration by moving, relative to one another, the charge-coupleddevice and the detection zone such that the charge coupled device trackslight signals emitted from the markers.
 18. The apparatus of claim 17,wherein said detection zone is located along said channels.
 19. Theapparatus of claim 17, wherein said detection zone is located outside ofsaid channels.
 20. The apparatus of claim 17, including at least fourco-extensive channels.
 21. The apparatus of claim 17, further comprisingmodulating optics disposed along an optical path extending between saidCCD and said detection zone.
 22. The apparatus of claim 17, wherein saidlight source is a light-emitting diode.
 23. An apparatus for detectinganalytes, comprising: a plurality of analyte-migration lanes; a lightsource; an excitation-light pathway extending from said light source tosaid analyte-migration lanes, with said light source being disposed todirect light along said excitation-light pathway; a charge-coupleddevice configured to operate in time-delay integration mode andconfigured to receive light signals from the plurality ofanalyte-migration lanes; and a fluorescence-emission pathway opticallycoupling said analyte-migration lanes and said charge-coupled device;modulating optics disposed between the plurality of analyte-migrationlanes and the charge-coupled device; and a system coupled to the chargecoupled device for effecting time delay integration by moving one ormore of the analyte-migration lanes, the modulating optics, and thecharge-coupled device.
 24. The apparatus of claim 23, including at leastfour co-extensive analyte-migration lanes.
 25. The apparatus of claim23, further comprising modulating optics disposed along one or both ofsaid excitation-light and fluorescence-emission pathways.
 26. Theapparatus of claim 23, wherein said light source is a light-emittingdiode.
 27. An apparatus for detecting analytes in a sample, comprising:one or more channels including one or more detection zones; one or moreirradiation sources, each irradiation source being capable ofirradiating with radiation at least one of the one or more detectionzones; a detector array disposed for collecting light signals emittedfrom the one or more detection zones by markers excited by theradiation, the detector array being capable of generating one or moreoutput signals; and a charge-shifting system coupled to the detectorarray for effecting time delay integration of charges on the detectorarray which correspond to the light signals, the charge-shifting systembeing capable of accumulating the charges on the detector array andmoving the detector array at a speed substantially synchronized torespective speeds of migration of bands of markers that migrate acrossthe one or more detection zones, to track an emission signal.
 28. Theapparatus of claim 27, wherein the charge-shifting system is capable ofsynchronizing prior to the detector array generating the one or moreoutput signals.
 29. An apparatus for detecting analytes in a sample,comprising: a plurality of channels, each channel including one or moredetection zones; one or more irradiation sources, each irradiationsource being capable of irradiating with radiation at least one of theone or more detection zones; a detector array disposed for collectinglight signals emitted from the detection zone by markers excited by theradiation, the detector array being capable of generating one or moreoutput signals; masks to selectively mask the plurality of channels suchthat light signals from each of the plurality of channels are keptseparate from light signals from others of the plurality of channels;and a system coupled to the detector array for effecting time delayintegration of the charges on the detector array which correspond to thelight signals by accumulating the charges before reading the charges atthe output of the detector array, the system being capable of effectingtime delay integration by moving, relative to one another, the detectorarray and light signals from the detection zone, the system comprising acharge-shifting system capable of shifting accumulated chargescorresponding to light signals emitted from the one or more detectionzones.
 30. The apparatus of claim 29, wherein the detector arraycomprises a CCD.
 31. The apparatus of claim 29, wherein the system foreffecting time delay integration moves one or more of the detectionzone, the modulating optics, and the detector.
 32. The apparatus ofclaim 29, wherein the charge-shifting system is capable of shiftingaccumulated charges corresponding to light signals emitted from the oneor more detection zones at a speed substantially synchronized to speedsof migration of bands of markers that migrate across the one or moredetection zones.